Senescence-associated ultrastructural features of long-term cultures of induced pluripotent stem cells (iPSCs)

Abstract

Induced pluripotent stem cells (iPSCs) hold great promise for developing personalized regenerative medicine, however characterization of their biological features is still incomplete. Moreover, changes occurring in long-term cultured iPSCs have been reported, suggesting these as a model of cellular aging. For this reason, we addressed the ultrastructural characterization of iPSCs, with a focus on possible time-dependent changes, involving specific cell compartments. To this aim, we comparatively analysed cultures at different timepoints, by an innovative electron microscopic technology (FIB/SEM). We observed progressive loss of cell-to-cell contacts, associated with increased occurrence of exosomes. Mitochondria gradually increased, while acquiring an elongated shape, with well-developed cristae. Such mitochondrial maturation was accompanied by their turnover, as assessed by the presence of autophagomes (undetectable in young iPSCs), some containing recognizable mitochondria. This finding was especially frequent in middle-aged iPSCs, while being occasional in aged cells, suggesting early autophagic activation followed by a decreased efficiency of the process with culturing time. Accordingly, confocal microscopy showed age-dependent alterations to the expression and distribution of autophagic markers. Interestingly, responsivity to rapamycin, highest in young iPSCs, was almost lost in aged cells. Overall, our results strongly support long-term cultured iPSCs as a model for studying relevant aspects of cellular senescence, involving intercellular communication, energy metabolism, and autophagy.

Mitochondrial and Peroxisomal Alterations Contribute to Energy Dysmetabolism in Riboflavin Transporter Deficiency

“…For focused ion beam/scanning electron microscopic (FIB/SEM) analyses, cells were plated and treated in a chamber slide (Lab-Tek™ II Chamber Slide System, ThermoFisher Scientific) and fixed in 0.5% glutaraldehyde and 2% paraformaldehyde, in 0.1 M cacodylate buffer, pH 7.4, for 1 h, at 4°C. Postfixation with osmium tetroxide, dehydration, and embedding in epoxy resin were performed as previously described [ 45 ]. Resin-embedded cells were mounted on stubs using a self-adhesive carbon disk and gold sputtered by an Emitech K550.…”

Mitochondrial Abnormalities in Induced Pluripotent Stem Cells-Derived Motor Neurons from Patients with Riboflavin Transporter Deficiency

“…Treated and untreated cells were fixed and embedded in epoxy resin, according to Colasuonno and coll. [ 11 ]. Electron microscopic observations and analyses were performed by a DualBeam focused ion beam/scanning electron microscopy (FIB/SEM, Helios Nanolab, FEI, Hillsboro, OR, USA).…”

Mitochondrial Abnormalities in iPSC-Derived Motor Neurons From Patients With Riboflavin Transporter Deficiency

“…Ultrastructural results well correlate with confocal data. Indeed, FIB/SEM analysis shows in RTD MNs remarkable mitochondrial dysmorphologies, strongly suggesting their dysfunction [9,11]. Altered features associated to RTD condition include disrupted mitochondrial cristae and the presence of vacuoles in mitochondrial matrix.…”

“…Treated and untreated cells were fixed and embedded in epoxy resin according to Colasuonno and coll. [11]. Electron microscopic observations and analyses were performed by a DualBeam Focused Ion Beam/Scanning Electron Microscope (FIB/SEM, Helios Nanolab, FEI, Hillsboro, OR, USA).…”